The glycoprotein transferrin (Tf) is the primary iron-transport protein in human plasma. Tf plays an important role in directing plasma iron to bone marrow for incorporation into developing reticulocytes. Tf consists of a single polypeptide chain of 679 amino acids with two potential sites for glycosylation, and has a molecular weight of 79,570 Daltons.
Human Tf is microheterogenous with respect to its glycosylation. Nine glycoforms of Tf, referred to as sialoforms, can exist, varying in the number of sialic acid residues bound to the surface glycans. The tetrasialoform is found at the highest concentration in normal human serum, with the hexa-, penta-, and tri-sialoforms detectable at lower concentrations. The di-, mono-, and asialoforms, collectively referred to as "carbohydrate-deficient transferring" (CDT's) are observed only under certain pathological conditions, including patients chronically consuming excessive amounts of alcohol and in patients having "carbohydrate-deficient glycoprotein syndrome (CDGS)," which is manifested by severe neurological deficiencies.
Capillary electrophoresis (CE) is a technology useful for the analysis of a variety of analytes of basic scientific and clinical importance. CE involves the electrophoretic separation of analytes, typically in narrow-bore fused silica capillaries. The capillaries employed in CE provide a high surface-to-volume ratio which allows for very efficient dissipation of Joule heat generated from large applied fields. Other techniques operating on this principle are known in the art, such as the use of capillary-like microchannels or troughs etched in glass chips or other materials to form microfabricated devices. As used herein, the term "capillary" refers to such microchannels or troughs in such devices, and the term "capillary electrophoresis," or "CE," refers to techniques and systems employing such microchannels, troughs, or devices.
Electrophoretic separations can be easily performed at up to 30,000 volts while the capillary remains close to ambient temperature. This permits "minute" time-scale separations and a significant reduction in analysis time in comparison to slab gel electrophoresis. Moreover, it is well established that both the speed of the separation as well as the resolution of the components are a direct function of the applied electric field in electrophoresis. Using applied electric fields up to 100 times those typically used with conventional electrophoretic methods, CE usually outperforms slab gel electrophoresis, not only in speed of analysis, but also in resolution.
In addition, the microliter column volume (e.g., 0.92 .mu.l for a 47 cm.times.50 .mu.m capillary) used in CE provides other advantages including relatively low amounts of reagent and sample consumed and the speed at which the capillary can be regenerated for subsequent analyses.
CE thus provides rapid (minute time-scale), high efficiency (10.sup.5 -10.sup.7 theoretical plates/m), reproducible (relative standard deviation&lt;1%), automated separations on low volume samples under native or denaturing conditions, all of which provide distinct advantages over conventional slab gel electrophoresis.
A number of modes of CE have developed in the art, including capillary zone electrophoresis (CZE; separation in low ionic strength buffer), capillary isoelectric focusing (CIEF; separation of proteins in a pH gradient of ampholines) and capillary gel electrophoresis (CGE; separation in a polymeric sieving matrix).
Although CE provides efficient, reproducible and automated separation, the successful and reproducible CE separation of proteins, and especially of protein isoforms having virtually identical molecular weights, is problematic. Proteins have an intrinsic tendency to interact not only with other proteins, but also with the capillary wall. Certain proteins have been successfully separated using CE, but there is no universal approach or set of conditions applicable to all target proteins. A CE system able to separate a target protein from other components in a sample solution or to resolve clinically significant isoforms of a target protein within a sample solution would be an attractive alternative to less efficient and reliable methods in developing an assay for such protein or proteins.
Analysis of human Tf by CIEF and CZE has been reported in the literature. Each of these approaches, however, has disadvantages in the context of achieving a useful clinical assay. CZE relies solely on mass-to-charge ratios for separation and, therefore, slight differences in mass or charge may not be adequate for resolution. CIEF can effectively separate Tf isoforms based on differences in their individual pI's, but separation takes too long to be a viable means of efficiently handling a large number of samples.
Methods currently available in the clinical setting for discriminating Tf glycoforms include isoelectric focusing in slab gels followed by visualization of immunofixed glycoforms, immunopurification followed by isoelectric focusing and protein staining, and anion exchange with immunological detection of subfractions of Tf's at precisely controlled pH (5.65). In the latter method, CDT's are poorly retained by the column and are separately measured by radioimmunoassay (RIA).
While these methods function adequately for the analysis of serum Tf, they would be prohibitively expensive (reagent and labor costs) for handling a large number of samples in a high throughput manner (e.g., several hundred per day) and are not amenable to automation.
There is currently no automated assay available for the diagnosis of chronic alcoholism or CDG syndrome or for the resolution of Tf glycoforms generally. It would be highly advantageous to provide a system for resolving Tf glycoforms that would be amendable to automation and be capable of rapidly analyzing samples in a high throughput manner. The desirable assay would be capable of rapidly detecting Tf glycoforms indicative of pathologies characterized by the presence of CDT's.